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Abstract

Background

The fungal pathogen Fusarium graminearum causes Fusarium Head Blight (FHB) disease on wheat which can lead to trichothecene
mycotoxin (e.g. deoxynivalenol, DON) contamination of grain, harmful to mammalian health. DON is
produced at low levels under standard culture conditions when compared to plant infection
but specific polyamines (e.g. putrescine and agmatine) and amino acids (e.g. arginine and ornithine) are potent inducers of DON by F. graminearum in axenic culture. Currently, host factors that promote mycotoxin synthesis during
FHB are unknown, but plant derived polyamines could contribute to DON induction in
infected heads. However, the temporal and spatial accumulation of polyamines and amino
acids in relation to that of DON has not been studied.

Results

Following inoculation of susceptible wheat heads by F. graminearum, DON accumulation was detected at two days after inoculation. The accumulation of
putrescine was detected as early as one day following inoculation while arginine and
cadaverine were also produced at three and four days post-inoculation. Transcripts
of ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), two key biosynthetic
enzymes for putrescine biosynthesis, were also strongly induced in heads at two days
after inoculation. These results indicated that elicitation of the polyamine biosynthetic
pathway is an early response to FHB. Transcripts for genes encoding enzymes acting
upstream in the polyamine biosynthetic pathway as well as those of ODC and ADC, and
putrescine levels were also induced in the rachis, a flower organ supporting DON production
and an important route for pathogen colonisation during FHB. A survey of 24 wheat
genotypes with varying responses to FHB showed putrescine induction is a general response
to inoculation and no correlation was observed between the accumulation of putrescine
and infection or DON accumulation.

Conclusions

The activation of the polyamine biosynthetic pathway and putrescine in infected heads
prior to detectable DON accumulation is consistent with a model where the pathogen
exploits the generic host stress response of polyamine synthesis as a cue for production
of trichothecene mycotoxins during FHB disease. However, it is likely that this mechanism
is complicated by other factors contributing to resistance and susceptibility in diverse
wheat genetic backgrounds.

Background

Fusarium head blight (FHB) or scab is one of the most important diseases of wheat and other
small grain cereals in many wheat growing regions [1]. FHB is caused mainly by F. graminearum, but also by other related Fusaria. A characteristic of FHB disease is the production of trichothecene mycotoxins such
as deoxynivalenol (DON) by the fungus in infected heads. Trichothecenes are phytotoxic
[2] and their biosynthesis during FHB is necessary for full pathogen virulence, and the
spread of the fungus through the infected wheat head [3-5]. Importantly, trichothecenes such as DON are toxic to animals and humans when present
in feed and food products, respectively [6]. Because of the undesirable effects of DON on human and animal health, its presence
in grain is tightly regulated in many wheat grain markets [7]. Consequently, there is also a strong interest in the development of novel technologies
for reducing DON levels in infected wheat either through breeding or via chemical
and/or biological control methods [8,9].

One constraint on our ability to reduce DON production by Fusarium pathogens has been a limited understanding of environmental and host-associated genetic
factors that regulate the production of trichothecene toxins during the infection
process [10]. It is well known that the amount of DON produced by F. graminearum during the infection of living wheat plants is much higher than that observed under
common culture conditions, including growth on autoclaved wheat grains. This suggests
that specific host factors stimulate DON production during the infection process [11,12]. Several factors that may induce the production of DON by F. graminearum during the infection process have been proposed. These factors include hydrogen peroxide
[13], sugars [14], acidic pH [15,16], and fungicides [17]. In particular, we have recently shown in a screen of various nitrogen containing
compounds that the most potent inducers of DON production by F. graminearum were metabolites (e.g. arginine, ornithine, agmatine, citrulline and putrescine) of the plant polyamine
biosynthetic pathway shown in Figure 1[18]. These metabolites of the polyamine pathway appear far more potent than hydrogen
peroxide and sugars at inducing DON production in vitro [18]. Importantly, the levels of DON produced in culture filtrates of F. graminearum after growth in the presence of inducing amines such as agmatine, putrescine and ornithine
were extremely high (> 500 ppm) [18]. The primary biosynthetic enzyme in trichothecene biosynthesis in F. graminearum is trichodiene synthase [19] encoded by TRI5 [5] and transcripts of TRI5 were also induced in the fungus by polyamines in culture to levels equivalent to those
observed in infected heads [18]. Interestingly, both spermine and spermidine, two very common and abundant plant
polyamines, did not induce DON production, suggesting that not all polyamines were
inducers of DON [18]. Nevertheless, the stimulation of trichothecene biosynthesis by specific polyamines
appears to be a general response in Fusarium pathogens because the DON-inducing polyamine agmatine was also able to induce the
production of T-2 toxin, another trichothecene, in F. sporotrichioides [18]. Because of the potency of polyamine inducers in stimulating trichothecene mycotoxins
in culture, Gardiner et al. [18] hypothesised that the pathogen may perceive polyamines and related amino acids as
cues for the production of toxins during the infection process.

Figure 1.Principle pathway of polyamine biosynthesis in plants. Expression of all genes encoding enzymes of the pathway were investigated except
for agmatine deaminase, carbamoylputrescine amidase and spermine synthase. Metabolites
measured in this study included arginine, putrescine, spermine and spermidine. Metabolites
indicated by an asterisk were previously shown to have in vitro trichothecene inducing activity [18].

Polyamines are well known as metabolites rapidly induced by diverse abiotic stresses
in plants, including salinity, drought, chilling, hypoxia, ozone, heavy metals and
UV irradiation [20,21]. The biosynthetic pathway of the principle polyamines of plants is well understood
(Figure 1). Two routes of synthesis to the primary amine putrescine have been described with
the first steps being decarboxylation of either ornithine or arginine catalysed by
ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), respectively. In subsequent
reactions aminopropyl groups are generated from S-adenosylmethionine (SAM) by SAM decarboxylase to convert putrescine to spermidine
and subsequently spermine. Cadaverine is thought to be produced by the decarboxylation
of lysine catalysed by lysine decarboxylase in prokaryotes but in eukaryotes this
step is less well defined [22,23]. A role for polyamines in protection against the stress-induced cellular damage has
been demonstrated in transgenic plants of rice, Arabidopsis, tobacco, tomato and pears
that accumulate high levels of polyamines through the over-expression of key biosynthetic
enzymes in the polyamine biosynthetic pathway [reviewed by [24]].

In contrast to abiotic stresses where polyamine accumulation is a general stress response,
the response of polyamines to pathogen challenge appears to be more dependent on the
host-pathogen system under study. Increases in polyamine content in susceptible barley
during rust and powdery mildew disease development [reviewed by [25]] and in rice after infection with the blast pathogen Magnaporthe grisea [26,27] have been reported. In contrast, infection of tobacco with powdery mildew and downy
mildew pathogens as well as the necrotroph Alternaria tenuis led to decreased levels of polyamines [28]. Increasing polyamine levels via over-expression of polyamine biosynthetic enzymes
has been shown to increase the tolerance of tobacco to F. oxysporum [29]. Conjugates of agmatine, such as hordatine from barley and feruloylagmatine from
wheat, are also known to have antifungal activity [30,31]. Furthermore, polyamine biosynthesis appears to be positively regulated by the plant
defence hormone methyl jasmonate in wheat and barley but not in rice [32-34]. Polyamine oxidases are also thought to contribute to reactive oxygen species generation
during defence against diverse pathogens [26].

Because polyamines are generically induced during plant stress, it is possible that
Fusarium pathogens have evolved to recognise these metabolites during the infection of wheat
plants to trigger toxin production. However, it is currently unknown what polyamines
or related metabolites accumulate during FHB development. Gardiner et al [18] described a preliminary experiment that suggested that putrescine was elevated in
wheat heads during FHB disease development, but only one metabolite and one post-inoculation
time-point was analysed. In the present study, we have studied the expression of wheat
genes that encode polyamine biosynthetic enzymes during FHB development to determine
if this host pathway is activated during infection. We have also analysed a spectrum
of polyamines and amino acids at multiple time-points after inoculation and across
multiple bread wheat genotypes. These experiments have permitted a comparison of the
temporal and quantitative patterns of accumulation of polyamines and amino acids during
infection with the timing and concentrations of DON in infected heads. The results
demonstrate that the core polyamine biosynthetic pathway is activated early on during
F. graminearum infection in wheat and that putrescine accumulation occurs prior to toxin production
by the pathogen. This latter observation provides additional support to the view that
polyamines are not only inducers of toxin production by the pathogen in vitro but may also play a similar role in planta.

Results

Polyamine, amino acid and DON accumulation during FHB disease development

The observation that intermediates of the polyamine pathway are strong inducers of
TRI5 expression and DON production by F. graminearum in culture led us to investigate the concentrations of these compounds in wheat heads
during infection. Inflorescences of a susceptible wheat cultivar were spray inoculated
with conidia of F. graminearum at mid-anthesis and polyamines and free amino acids were analysed in head samples
taken daily over a seven-day period.

The putrescine concentration in the infected spikes increased rapidly and was almost
two fold that of the mock inoculated control at three days post-inoculation and then
reached a plateau at approximately 500 nmoles/g fresh weight (Figure 2A). Concentrations of spermidine increased more slowly and appeared to be higher than
mock inoculated controls after four to seven days post-inoculation. Spermidine was
the most abundant polyamine reaching a level of 1400 nmoles/g fresh weight. Spermine
levels were more variable and no significant differences were observed in its concentration
in infected relative to mock-inoculated heads (Figure 2C). The levels of putrescine and spermidine increased during the seven-day period even
in the mock inoculated heads, indicating that polyamine accumulation may be a normal
part of development (Figure 2A-B). Similar increases in these polyamines were observed during the early stages of
grain development in field grown wheat [35] and also during development of the rice panicle [36]. Putrescine is an inducer of DON in vitro and it was interesting that inoculation led to a rapid increase in putrescine levels
which were significantly higher than those of mock-inoculated controls at one, two
and three days post-inoculation (Figure 2A). Agmatine is also a potent DON inducer and a precursor of putrescine synthesis but
using our extraction methods we were unable to detect agmatine in wheat heads (data
not shown). This is most likely due to the instability of agmatine, as observed by
others as well [37]. Interestingly, another polyamine and potent inducer of DON production, cadaverine
[15], was not detected in the mock-inoculated heads at all but accumulated in infected
heads, with significant increases observed at three days post-inoculation, eventually
reaching a concentration of 200 nmoles/g fresh weight. Quantitative RT-PCR measurement
of fungal polyamine biosynthetic gene expression indicated a lack of induction of
fungal transcripts relative to those of the host during infection (data not shown)
and coupled with plant material being the dominant contributor to biomass at all time
points, this suggests that the measured polyamines are most likely almost exclusively
of plant origin.

We also analysed the amino acid content of these heads because arginine was a strong
DON inducer while some other amino acids such as lysine, methionine and phenylalanine
were weak DON inducers [18]. In addition, combinations of amino acids and polyamines (e.g. methionine with putrescine)
appeared to act synergistically for DON induction in culture [18]. Unlike polyamines, most amino acids did not show any increase in concentration during
the development of the head in mock-inoculated controls. Some amino acids did increase
in concentration during FHB disease development and these included glycine, valine,
arginine, alanine, phenylalanine, lysine and leucine as well as threonine and/or citrulline
which could not be resolved (Figure 3 and Additional File 1). However, a significant increase in the concentration of most of these compounds,
and particularly the potent DON inducer arginine, was only observed later in the infection
time-course (Figure 3). The concentration of arginine increased from approximately 1 μmol/g fresh weight
at one day post-inoculation to up to 7 μmol/g fresh weight at seven day post-inoculation.
The only amino acid that rapidly responded to inoculation was isoleucine where an
increase was observed at one day post-inoculation but this was not sustained at later
stages of infection (Additional File 1). Ornithine is another potent DON inducer [18] but this amino acid was below the detection level in wheat heads using our instrumentation.
Using more sensitive equipment (Waters AccQ-Tag Ultra at the Australian Proteome Analysis
Facility), we were able to detect trace amounts of ornithine (maximum observed 0.05
μmol/g fresh wt).

In order to temporally compare the accumulation of potential DON inducing polyamines
and amino acids measured in the time-course experiment (Figure 2 and Figure 3) with disease and DON levels, we also measured DON levels and fungal biomass (Figure
4) during disease development. DON was not detectable in the infected heads until three
days post-inoculation and then steadily increased, reaching 1200 ppm in fresh head
tissue at seven days post-inoculation. Fungal biomass in infected tissue was measured
by quantitative PCR of a fungal DNA sequence relative to that of a plant sequence
in DNA extracted from infected heads. Similarly, only minor increases in fungal biomass
for the first two days post-inoculation was detectable but this increased rapidly
thereafter and peaked at five to six days after inoculation. Therefore, these experiments
suggest that the induction of putrescine in infected heads preceded the production
of DON in the fungus.

Induction of genes encoding polyamine biosynthetic enzymes during FHB

The biosynthesis of putrescine, spermine and spermidine from primary amino acid metabolism
involves several enzymatic steps, with two potential branches of putrescine synthesis
from arginine via either ornithine or agmatine as intermediates (Figure 1). We were interested in knowing which genes of this pathway might be activated by
fungal infection and the timing of this response. To determine this, we first identified
wheat homologues for 11 of the polyamine biosynthetic enzymes (Figure 1 & Table 1). We used previously annotated rice polyamine sequences as queries in searches of
wheat expressed sequence tag clusters available in the WhETS database. Although not
a definitive analysis of copy number, based on the clusters produced by WhETS for
the query sequences all polyamine pathway genes described in this manuscript appear
to be present in single copies in each of the three wheat homoeologous genomes. The
wheat sequences identified in these analyses were used to design primers (Table 1) for Quantitative Real-Time Reverse Transcriptase PCR analysis of transcripts in
RNA samples from mock- and F. graminearum-inoculated heads of the susceptible wheat cultivar Kennedy during FHB disease development.

Of the 11 transcripts studied, only two transcripts encoding a wheat orthologue of
ornithine decarboxylase (ODC) and an isoform of arginine decarboxylase designated
as ADC2 (see below) showed a significant increase following inoculation with F. graminearum (Figure 5). At one day post inoculation, ODC was induced seven fold compared to mock albeit
with some variability (P = 0.054). Transcripts for ODC were significantly induced at two days post-inoculation (~40-fold controls, P = 0.003) and continued to increase up to three days post-inoculation where a level
>100-fold that of mock controls was reached and maintained for the seven day duration
of the experiment.

Figure 5.Expression of genes of the polyamine biosynthesis pathway during Fusarium head blight
infection. Values are the relative expression between infected plants compared to mock inoculated
plants. Error bars are the standard error of the mean. n = 4 individual heads. Asterisks
indicate statistically significant (t-test P < 0.05) differences between infected and mock treated samples at that time point.

In the model plant Arabidopsis, two genes designated as ADC1 and ADC2 encode separate isoforms of ADC. ADC1 and ADC2 are functionally redundant as mutants for either gene are viable but double mutants
are not [38]. Interestingly, ADC2, but not ADC1, has been shown to be responsive to salt and other abiotic stresses in Arabidopsis
[39,40]. Similarly, we identified two distinct ADC sequences in wheat. However, based on nucleotide and amino acid comparisons between
Arabidopsis and cereal ADC sequences, which one of these sequences is the direct wheat
orthologue of the Arabidopsis ADC2 gene could not be established. Of these two sequences, only one was inducible during
FHB disease development (Figure 5A and data not shown), suggesting that similarly to Arabidopsis, different ADC isoforms are differentially regulated under stress in wheat. We therefore designated
the pathogen-inducible wheat ADC gene as ADC2. This gene was induced three-fold and ~10-fold, relative to mock-inoculated controls,
at two and four days post-inoculation (Figure 5A).

To further characterize the relative speed of induction we compared the expression
patterns of ODC and ADC2 to those of the peroxidase encoding gene TaPERO, which is known to be transcriptionally induced in wheat following inoculation by
F. graminearum, F. culmorum or F. pseudograminearum [41,42]. TaPERO was induced similarly to ODC and ADC2 with three-fold induction observed at one day post-inoculation, followed by a rapid
increase between two and three days post-inoculation relative to mock-inoculated controls
(Figure 5C). This is consistent with ODC and ADC2 induction being part of the coordinated defence response to FHB.

The polyamine pathway is co-ordinately regulated in the rachis under infection

It is now well known that one of the roles that DON plays during FHB development is
to allow the fungus to colonise the rachis of infected spikelets and spread into the
rachis of the spike [4,43]. Additionally, our previous work has demonstrated TRI5 is strongly expressed in the rachis tissue of wheat [18]. Given the importance of rachis in disease spread within the infected head, we were
particularly interested in knowing whether the polyamine pathway is specifically or
especially induced in this tissue during FHB development. To test this, we collected
the rachis and the spikelets separately from spray- and mock-inoculated heads at six
day post-inoculation. Putrescine quantification and quantitative RT-PCR was then carried
out on each material. In mock-inoculated heads, putrescine levels were not significantly
different in the rachis and spikelets (Figure 6). However, under infection, levels of putrescine in the rachis increased more dramatically
than those in the spikelets (Figure 6; difference between infected rachis and spikelets P = 0.011, mock versus infected for both spikelets and rachis P < 0.001). Similar levels of induction of ODC and ADC2 were observed in both rachis and spikelets. However, transcripts encoding for argininosuccinate
synthase (P = 2×10-4) and to a lesser extent argininosuccinate lyase (P = 0.04) and orthinine carbamoyl transferase (P = 0.001) all showed significantly higher levels of induction in rachis material (Figure
7). ASS, ASC and OCT showed no significant transcriptional induction in whole head samples. The enzymes
encoded by these genes catalyse earlier steps in the polyamine biosynthetic pathway
than ODC and ADC (Figure 1) suggesting a more coordinated induction of the pathway may occur in the rachis following
infection.

Polyamine induction is a general response of resistant and susceptible wheats to FHB

To test whether activation of the polyamine biosynthetic pathway by F. graminearum correlates with resistance or susceptibility to FHB, we investigated the responses
of 24 diverse bread wheat lines that have been reported to vary in their response
to FHB [44]. As described in the Material and Methods, we first confirmed in independent inoculation
experiments that these 24 lines were indeed different in their response in FHB disease
symptom development and DON accumulation. Overall, there was a good correlation (r
= 0.75) between DON levels and disease susceptibility across these lines, supporting
the already known link between DON and disease symptom development in these lines.
These lines were also inoculated and whole heads sampled at three days post-inoculation
and analysed for polyamine and amino acids and the data for putrescine is shown in
Figure 8. Considerable variation in the levels of putrescine, from 150 to 732 nmoles/g fresh
weight in mock and from 318 to 1130 nmoles/g fresh weight in inoculated heads, was
evident across the genotypes (Figure 8). As described earlier for cv. Kennedy (Figure 2), we found a significant induction of putrescine production in 22 of the wheat lines
tested with the exception of Soba komugi 1C and Synthetic-W7984 (Figure 8). Similar inductions were observed for spermidine in all the lines examined (Additional
File 2). Cadaverine detection (data not shown) and spermine levels (Additional File 2) were variable across the genotypes. No amino acid, including the potent DON inducer
arginine showed any induction upon infection at three days post inoculation in these
lines (data not shown). These results, therefore, indicate that the induction of putrescine
and spermidine is a general response of wheat to infection and occurs in both resistant
and susceptible lines.

Figure 8.Putrescine levels in mock and inoculated wheat heads of diverse wheat cultivars. Measurements were taken at three days post inoculation. Error bars are the standard
error of the mean n≥4. Genotypes are plotted in increasing levels of DON as described
in the Materials and Methods.

Additional file 2.Spermidine and spermine concentrations in mock- and Fusarium head blight infected
diverse wheat lines. Spermidine (A) and spermine (B) concentrations in mock- and Fusarium head blight
infected diverse wheat lines. Error bars are the standard error of the mean n≥4. Genotypes
are plotted in increasing order of DON concentration.

We tested the correlations between putrescine and spermidine levels found in both
mock and inoculated heads and FHB disease ratings and DON levels in these wheat lines
(see Material and Methods) but no significant correlation was found (data not shown).
These results suggest that polyamine induction is a general response of both FHB resistant
and susceptible wheats to infection.

Discussion

Several factors are known to influence the production of trichothecenes by F. graminearum in culture [13-15,18]. However, whether these factors also affect toxin production in planta during pathogenesis is unknown. The plant stress metabolites polyamines are one of
the most potent toxin inducing factors in culture and result in toxin and biosynthetic
enzyme mRNA levels equivalent to those observed in infected heads [18]. Here we have tested whether it is plausible that polyamines may act as in planta inducers of trichothecene production in wheat. By analysing the temporal changes in
polyamine levels and the expression of the genes for their biosynthesis in relation
to the biosynthesis of the trichothecene DON during FHB disease development. A key
finding was that the induction of expression of key polyamine biosynthetic pathway
genes and increase in polyamine (e.g. putrescine) levels following inoculation of
wheat heads preceded the detection of DON in infected tissue. Induction of ODC by
FHB was also observed in publically available global expression data in both resistant
and susceptible near isogenic lines for Fhb1/fhb1 in wheat, and to a lesser extent
during barley FHB [45-47]. Furthermore the timing of induction of ADC2 and ODC was similar to that of TaPERO known to be induced in response to Fusarium spp. [41,42]. These observations are consistent with a model where the early stages of fungal
challenge of wheat flowers induce polyamine biosynthesis as a generic host stress
response and this response is sensed by the pathogen as a cue for boosting trichothecene
mycotoxin production such as DON.

It is well established that DON production in the fungus is not required for the initial
infection of wheat flowers. However, DON is required as a virulence factor facilitating
fungal colonisation and disease spread from the initially infected floret to other
florets via the rachis [4,48]. In the absence of fungal DON production, the progress of F. graminearum is halted at the rachis node by plant cell wall thickenings as part of the normal
defence response [4]. The model proposed above where the fungus initially stimulates a stress response
in the host and then uses this as a trigger for DON production is also consistent
with the proposed role for DON later in the infection process rather than it being
necessary for initial infection processes.

The importance of the rachis node for induction of DON production in planta has recently been elegantly demonstrated [43]. The tight tissue specificity for DON production observed in the rachis tissue suggests
that any DON-inducing factors are likely to be preferentially synthesised in this
zone. Interestingly, Peeters et al. [35] detected only putrescine and no other polyamines in the rachis during wheat anthesis,
suggesting some tissue specificity in the production of this particular polyamine
that is also a potent inducer of DON production. In our analysis of transcripts for
polyamine biosynthesis from whole heads we observed significant induction of only
ODC and ADC2 but when the rachis was sampled separately there was also a significant induction
of transcripts for three other enzymes earlier in the pathway suggesting a coordinated
induction of the biosynthetic steps to putrescine. This is also consistent with the
model proposed for putrescine as an inducer of DON production during infection.

The concentration of putrescine reached in heads following inoculation was approximately
0.5 mM based on a sample fresh weight basis. In culture, the lower concentration limit
observed for the induction of DON by polyamine inducers such as putrescine is approximately
1 mM (data not shown) which is higher than the putrescine concentrations measured
here in planta. Although this may argue against a possible in planta DON-inducing role for this metabolite, it should be noted that the actual putrescine
concentration at the host-pathogen interface may be higher than the overall tissue
level and it is possible that DON is synergistically induced by multiple compounds
and conditions as demonstrated previously [15,18]. Also, it is possible that infection hyphae may differ in their sensitivity to inducing
compounds when compared to vegetative hyphae growing in batch axenic culture conditions.
More definitive information on the role of polyamines may be obtained using transgenic
plants silenced for ODC and/or ADC2 that contain significantly reduced levels of polyamines
in heads. However, given the central importance of polyamines in many biological processes
and the branched biosynthesis pathway in wheat, plants with reduced polyamine levels
maybe difficult to generate. Fungal mutants that are non-responsive to the polyamine
signals may also be useful to definitively test this hypothesis.

Our analysis of infected wheat heads could not discriminate between polyamines and
amino acids of fungal and plant origin. However, the early induction of polyamines
such as putrescine, before fungal biomass increases appreciably together with the
increases observed in transcripts of genes encoding plant polyamine biosynthetic enzymes
all provide evidence that the wheat plant is the major contributor of the metabolic
changes observed. Indeed PCR analysis of fungal polyamine biosynthetic genes showed
all genes analysed were constantly expressed during infection (data not shown). One
polyamine, cadaverine, was not detected in the non-inoculated heads and increased
in concentration in parallel with fungal biomass. Cadaverine is produced by decarboxylation
of lysine but no clear homologue of the bacterial lysine decarboxylase sequences were
identified in the genome sequence of F. graminearum (data not shown). In plants it is highly likely that decarboxylation of lysine is
a result of the ODC enzyme acting on lysine as an alternative substrate [49]. This may become significant when ODC transcripts, and presumably enzyme levels,
were so strongly induced by infection. This would explain why a delay is observed
in the production of cadaverine in infected tissue.

Conclusions

In summary, the in planta induction of DON biosynthesis in F. graminearum is a complex process to which polyamines are likely to contribute. Future work to
more specifically define the role of polyamines as inducers of DON during infection
will require functional tests with fungal mutants with impaired perception of polyamines
as well as tissue specific localisation of metabolites at the host-pathogen interface.

Methods

Plant material and inoculation

The susceptible Australian bread wheat (Triticum aestivum L.) cultivar Kennedy was used for all experiments unless indicated otherwise. Four
seeds were sown in 10 cm pots in potting mix amended with osmocote, and grown in a
controlled environment room with a photoperiod of 14 h, at 25°C and 50% relative humidity.
The light intensity was 500 mmol m-2 s-1. Night-time temperature was set at 15°C, with 90% relative humidity.

Unless specified otherwise, plants were spray inoculated at mid-anthesis with F. graminearum isolate CS3005 [50]. Approximately 2 mL of a 1×106 spores mL-1 or water for mock inoculations was applied to each head using a Preval Sprayer (Precision
Valve Corporation, NY, USA). Heads were covered with humidified plastic zip lock bags
following treatment. Plastic bags were replaced three days post-inoculation with a
glassine bag. All analyses were carried out on individual heads.

Polyamine and amino acid detection and quantification

Polyamines were quantified as previously described [18]. Free amino acids were extracted by grinding the heads under liquid nitrogen and
resuspending a known quantity (up to 500 mg) of the powder in 2 mL of methanol, incubated
at -20°C overnight and then 8 mL of water added and centrifuged to pellet solid matter.
500 μL of supernatant was dried down under vacuum. This extract was used with the
Waters (Milford, MA, USA) AccQ-Tag amino acid detection kit, with quantification by
HPLC according to the manufacturer's instructions. In a separate experiment, to allow
more sensitive quantification of ornithine, the amino acid analysis was performed
using the Waters AccQ-Tag Ultra chemistry at the Australian Proteome Analysis Facility
(Sydney, Australia).

Reverse transcriptase quantitative polymerase chain reaction

RT-qPCR was carried out as previously described [11]. To determine the target sequence of wheat polyamine genes for primer design, first
the rice locus encoding the target gene was identified by a combination of text querying
of putative function using the rice genome and reciprocal blast comparisons with the
relevant Arabidopsis loci. The rice loci numbers were then used to query the Wheat
Estimated Transcript Server (WhETS) database [52] to determine the orthologous wheat sequences. Where WhETS identified multiple homeologous
sequences, ClustalW [53] alignments were used to determine conserved regions of these sequences for design
of primers. Where suitable conserved regions could not be identified, multiple primer
pairs were utilised. Primers were designed using the Primer3 software [54]. Primer sequences used to amplify wheat polyamine genes and the orthologous rice
loci are listed in Table 1. Fungal biomass accumulation was estimated by comparing 18 S rRNA amplification from
the fungus and plant respectively using primers listed in Table 1. TRI5 gene expression was measured relative to fungal 18 S using primers listed in Table
1.

Deoxynivalenol quantification

Deoxynivalenol was quantified using the ELISA kit from Beacon Analytical Systems (Saco,
Maine, USA) as per the manufacturer's instructions.

Authors' contributions

DMG, KK, SP, FJT, JMM designed the research. DMG, SP and AR performed the experiments
and analysed data. DMG, KK and JMM wrote the manuscript. All authors read and approved
the manuscript.

Acknowledgements

We wish to thank Brendan Kidd and Johanna Bursle for excellent technical assistance.
We thank Dr Chunji Liu for providing seed for screening multiple wheat genotypes.
This work was jointly funded by CSIRO and Biogemma.